1. Objective 3.1 Background Several directed differentiation protocols have been proposed to facilitate the emergence of human HSPCs from iPSCs. These approaches aim at recapitulating hematopoietic development in vitro based on timed addition of cytokines and morphogens. Cells derived via directed differentiation display characteristics of primitive hematopoietic cells obtained from in-vivo sources, including expression of HSPC markers (e.g. CD34) and multilineage hematopoietic potential in clonogenic assays. However, despite recent promising reports, efficient derivation of functional transgene-free HSPCs with a robust capability for definitive in vivo engraftment and multilineage potential remains challenging. Current protocols are thought to recapitulate the primitive wave of hematopoiesis during development. In this wave, the yolk sac transiently gives rise to nucleated glycophorin A+ (CD235a+) red blood cells expressing embryonic hemoglobin, as well as primitive macrophages and megakaryocytes in the embryo. Only a later definitive wave of hematopoiesis, occurring intra-embryonically in the aorta-gonad-mesonephros (AGM) region in mammals and in particular the ventral wall of the dorsal aorta (DA), provides HSPCs with long-term repopulating potential. This wave is not dependably reproduced by current in vitro iPSC differentiation approaches. In addition, the scale-up of these protocols to generate sufficient cells for clinical transplantation has not been addressed. At present, most differentiation protocols are based on embryoid body (EB) formation or co-culture on OP9 cell lines. Translation of these iPSC-based approaches to a larger physiologic transplantation system would require liters of culture media and several grams of cytokines, rendering current approaches impractical and cost-ineffective for clinical applications. Therefore, strategies to advance the current state of de novo HSPC generation are needed. The generation of functional HSPCs from pluripotent stem cells will depend on the accurate recapitulation of embryonic hematopoiesis. Studies from different model organisms have shown that definitive HSPCs directly bud off from a progenitor population known as hemogenic endothelium (HE) in the dorsal aorta (DA) of the developing embryo through endothelial-to-hematopoietic transition (EHT). Using EB differentiation systems, Keller and colleagues have recently shown that HE is restricted to the CD34hi CD43- CD73mid CD184+ fraction and undergoes an EHT to generate RUNX1+ HSPCs with multilineage potential, perhaps the in vitro equivalent of the bona fide HE giving rise to functional HSPCs in the embryo. Hence, protocols for successful directed differentiation are contingent on reproducing the temporal waves of hematopoiesis during which the HE emerges and undergoes EHT for the generation of cells capable of long-term engraftment. Development and characterization of a novel protocol for hematopoietic differentiation of human iPSCs. In collaboration with Dr. Manfred Boehm (NIH), we developed a simple, monolayer-based, chemically-defined, scalable protocol for hematopoietic differentiation of human iPSCs. During the first 3 days, cells are induced toward mesoderm with morphogens and for the subsequent 9-18 days, cells are further differentiated into HSPCs with the addition of hematopoietic cytokines. Under these conditions, the cells rapidly form an adhesive monolayer composed of endothelial cells (days 3 to 5). Over the subsequent days, this monolayer undergoes an EHT as demonstrated by the emergence of round cells directly budding off the endothelial monolayer in time-lapse imaging studies. Importantly, these cells are released within the culture supernatant and can be easily harvested by simple pipetting. Flow cytometry analyses showed that the emerging round cells initially had a predominant CD45-CD235a+ phenotype, consistent with a primitive yolk-sac wave of hematopoiesis (days 5-10). From day 10, hematopoietic progenitors increased gradually with a maximum number (average 1.5x106 per 12-well plate) and percentage (average 43%) of CD45+CD34+ cells observed at day 12. These cells could form colonies in clonogenic progenitor assays. Strikingly, cells with a phenotype that enables the highest reported purity of human HSCs (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholo) were detected in the culture supernatant at days 10-12. However, these cells did not result in long-term engraftment in immuno-deficient (NSG) mice. To identify possible causes for the lack of durable repopulating potential of iPSC-derived HSPCs in this system, we further characterized the endothelial monolayer between day 5 and 12 of differentiation. We focused on recently described endothelial subsets within the CD34hiCD43- population, including the in vitro equivalent of definitive HE (CD34hi CD43- CD73- CD184- DLL4-), arterial vascular endothelium (CD34hi CD43- CD73mid CD184+), and venous vascular endothelium (CD34hi CD43- CD73hi CD184-). At each timepoint analyzed, definitive HE comprised <1% of the total cell population. Arterial vascular endothelium was also scarce, rising to a maximum of 6.6% at day 7 before declining to <1% by day 9 of culture. Venous vascular endothelium accounted for approximately 2% of total cells. Thus, our data suggest that the absence of a definitive HE population within a developing arterial niche, as occurs in the dorsal aorta (DA) during development, may explain in part the lack of engraftment potential of iPSC-derived HSPCs. To further understand the inability of iPSC-derived HSPCs to reconstitute a functional hematopoietic system, we investigated gene expression programs and master regulators that current iPSC differentiation protocols may fail to activate or silence. Recent advances now enable high-throughput sequencing of whole transcriptomes (RNA seq) at single cell resolution, allowing profiling of rare or heterogeneous populations of cells such as CD34+ HSPCs. We have derived several iPSC lines from HSPCs (CD45+ CD34+ CD38- cells) of 3 age- and sex-matched healthy individuals using an adapted Sendai viral reprogramming approach. iPSC lines from each donor were validated and differentiated using the monolayer-based protocol described above. Up to 40,000 iPSC-derived and bona fide CD45+CD34+ cells obtained from the same donors were profiled by single cell RNA-seq technology (Drop-Seq). A partial bioinformatic analysis of 30,000 bona fide and 15,000 iPSC-derived HSPCs from one donor has been completed. The existence of well-defined, non-overlapping clusters, as confirmed by tSNE maps, indicate a remarkably distinct global pattern of expression between somatic and iPSC-derived HSPCs consistent with the differences in engraftment between adult and iPSC-derived HSPCs. In-depth analysis is ongoing. We anticipate this approach will enable the rational design of novel enhanced iPSC differentiation methodologies. 2. Objective 3.2 In keeping with the central objective of this research program, the development of regenerative therapies for inherited blood disorders, we will derive molecularly reprogrammed iPSCs from available IBMFS specimens (e.g. FA, DBA) and/or culture available IBMFS patient-derived iPSC lines provided by collaborators (Dr. Mitchell Weiss-St. Jude Hospital, and Dr. Juan Carlos Izpisua Belmonte-Salk Institute). Targeted genetic correction of IBMFS lines will be performed in collaboration with Dr. Jizhong Zou (iPSC core facility, NHLBI). We will induce hematopoietic differentiation of genetically corrected patient-derived iPSCs using our optimized monolayer-based protocol. Finally, molecular, phenotypic and functional properties of HSPCs obtained from the differentiation cultures will be characterized.